The field of this invention is methods for determining ribonucleic acid.
There is substantial interest in being able to determine the occurrence and level of transcription in cells in vivo and in vitro. The transcriptional level is related to the strength of the promoter, the presence and amount of transcriptional factors, the level of binding of the transcriptional factors to the regulatory region, including the promoter and any enhancer, and the response of the cell to changes in its environment. Information concerning the occurrence of transcription and the level of mRNA produced can be associated with the pathways involved with the status of the cell, such as the type of cell, differentiation, maturation, response to internal and external changes, and the like. The information can be relevant to the effect of candidate drugs on the cell, the nature of the cell, as in metastatic cancer, active pathways in the cell, and other information of physiologic interest.
The amount of mRNA produced varies widely with the gene and the status of the cell. Frequently, the mRNA from the gene(s) of interest can be in very small amount, which can be further reduced by degradation by RNases, even when one quickly inactivates the RNases. In addition, since a single mRNA can be used as a template for the production of multiple copies of the encoded protein, very small amounts of mRNA may have profound effects on the physiology of the cell. In addition, one usually wishes to identify a small number of the total number of mRNAs that are present in the cell. In any system of amplification, there is always the concern that an mRNA that may be similar to the target(s) of interest may be present in much larger amount and becomes amplified. In this situation one will obtain a false negative, as the more abundant mRNA may obscure the detection of the less abundant mRNA. Methods of amplification should provide for high fidelity, so as minimize the opportunities for cross-reactivity with mRNAs other than the target mRNA(s).
There is also the fact that mRNAs have substantial secondary and tertiary structure. Unwinding the secondary and tertiary structure requires energy, so that the mRNA of interest may be less available to various methods of amplification, particularly isothermal amplification.
Numerous methods are found in the literature for detecting and amplifying mRNA, either as mRNA or cDNA. In many cases the methods require denaturation, so that one must use thermal cycling, which is inefficient. Where cDNA is used and the polymerase chain reaction is employed for amplification, not only is one concerned with thermal cycling, but the steps of reverse transcribing and amplification can introduce errors and the system is not useful for multiplexing. Other methods have used the Qxcex2 replicase, but the replicase is promiscuous and can and does produce copies of other than the target mRNA(s).
There is a need for methods that allow for multiplexing, so as to be able to amplify multiple mRNAs simultaneously without significant amplification of mRNAs other than the target mRNAs. Also, the method should permit high fidelity in copying the target mRNA and excluding other mRNAs. Desirably, the method should avoid thermal cycling and have a limited number of steps for amplification and identification of the target mRNAs and allow for a reasonable degree of quantitation. Other benefits would include a minimal number of reagents, stable reagents, exponential amplification, and ease of detection of the amplified product.
U.S. Pat. Nos. of interest include U.S. Pat. Nos. 4,725,537; 4,766,062; 4,795,701; 4,795,701; 4,957,858; 5,169,766; 5,385,834; 5,503,979; 5,620,851; 5,631,129; 5,916,779; 5,925,517; 6,037,130; 6,093,542; 6,100,024; 6,013,442; 6,132,997 and 6,180,338. Tm Bioscience Corp. (Toronto, Canada) sells hairpin capture probes as described in their brochures and on their web page. The T7 RNA polymerase is described in Sastry and Ross., Biochemistry (1997) 36:3133-44; Noren and Moreira, Book of Abstracts, 211th ACS National Meeting, New Orleans, La., Mar. 24-28 (1996); Cheetham, et al., Nature (London) (1999) 399:80-83; and Maslak, et al., Biochemistry (1993) 32:4270-4. Other references of interest include Lohse, et al., Proc. Natl. Acad. Sci. USA (1999) 96:11804-8; Phillips and Eberwine, Methods (1996) 10:283-8; Breaker, et al., Biochemistry (1994) 33:11980-6; and Milligan, et al., Nucleic Acids Res. (1987) 15:8783-98.
U.S. Pat. No. 6,025,133 describes hairpin probes as xe2x80x9cpromoter-sequesteredxe2x80x9d oligonucleosides to achieve xe2x80x9ctarget-triggeredxe2x80x9d amplification, which disclosure is specifically incorporated by reference in this application in its entirety.
Nucleic acid sequences are isothermally exponentially amplified using an RNA polymerase, a probe, a promoter initiator and RTPs. The probe comprises a masked promoter, and a double stranded nucleic acid region with a protruding sequence for binding to the target sequence to be amplified. Upon binding of the target to the protruding sequence, the target invades the double stranded region allowing the promoter initiator to bind to the masked promoter to provide a holopromoter initiating template dependent synthesis of RNA. Particularly, bulky groups are provided in proximity to the promoter region to inhibit transcription in the absence of target or target copy binding, particularly when a hairpin probe is used. The resulting RNA product can in turn act as the target nucleic acid invading probes and initiating additional copies of the target RNA.